Abstract Significance: New approaches for rapid identification and early preclinical validation of novel therapeutic targets are crucial to make important ?go/no-go? decisions and curb the cost of developing new cancer treatments. Genetically engineered mouse models (GEMMs) are a powerful platform to study disease initiation and maintenance, the tumor microenvironment and the responsiveness of cancers to known or novel therapeutics; however, the long lead times and high costs required to develop, intercross and maintain models with various cancer predisposing gene combinations have limited their practical utility in the drug discovery process. Recently, we have shown RNA interference (RNAi) in mice can serve as a fast alterative to gene deletion and be exploited experimentally to silence nearly any gene target, by the expression of synthetic short hairpin RNAs (shRNAs). Importantly, because it is reversible, gene silencing by RNAi better mimics the dynamics of small molecule inhibition than permanent genetic knockouts. Furthermore, with the advent of new genome editing techniques, such as CRISPR/Cas9 technology, we are able to introduce additional sensitizing lesions to induce disease pathogenesis. In synergy with RNAi technology, complex multi-allelic ESC based GEMMs can be generated without extensive intercrossing. Using this combination of CRISPR/Cas9 and RNAi technologies, we are able to not only model disease pathogenesis, but also mimic drug therapy in mice, giving us unprecedented capabilities to perform preclinical studies in vivo. Hypothesis: We hypothesize that CRISPR/Cas9-RNAi-GEMMs of cancer can be developed rapidly using new genome editing technologies (CRISPRs) to introduce additional sensitizing lesions and recombinase-mediated cassette exchange (RMCE) for precise integration of tetracycline inducible shRNAs to silence specific gene targets. Preliminary data: We have previously used CRISRP/Cas9 and RMCE to generate RNAi-GEMMs without any breeding. Specific Aims: As a proof-of-concept, we will develop a model of lung adenocarcinoma by using the CRISPR/Cas9 system to introduce a conditional KrasG12D allele into the endogenous locus and in situ delivery of sgRNAs targeting Trp53 which will be activated by a conditionally expressed Cas9 allele. We will further modulate mutant Kras or Mek1/2 activity by introducing tetracycline inducible shRNAs to model therapeutic inhibition. Finally, we will expand our flexible platform by producing validated, ?off-the-shelf? viral vectors carrying combination sgRNAs targeting commonly altered genes in NSCLC. Together, these studies will define a new paradigm and accelerate drug discovery research by creating a flexible platform for the generation of RNAi- GEMMs that will serve as innovative research tools, guiding the development of novel and effective therapeutics.